Optimization And Application Of The Method To Evaluate The Physiological Functions Of Prebiotics Fermentation Supernatant Based On Caco-2 Cell Model

Prebiotics mainly are kinds of carbohydrate that are not-easily digested in the upper gastrointestinal tract and contribute to human health by regulating the structure of gut microbiota.Prebiotics have been reported to improve many physiological functions such as catharsis,anticancer,promoting mineral absorption,regulating lipid metabolism and improving immunity.Currently,two different in vitro approaches are used to evaluate prebiotics:(i)in vitro fermentation model;(ii)in vitro cell model.Through the combination of these two models,we not only observe the structure change of fecal bacteria and their metabolites,but also reveal the physiological functions of the metabolites produced by fermentation.Nevertheless,there are few shortcomings for the in vitro cell model.Firstly,the existing determination method is unable to provide dynamic monitoring of cell reactions.Secondly,some key experimental factors such as loading volume and time of administration were unclear.To resolve the shortcomings with the in vitro cell model,a new technology platform-Real Time Cellular Analysis SP(RTCA SP)system was introduced in this study.Core of this system was the cell detection board integrated with microelectronic sensors at the bottom.Cell reactions was presented in the form of cell index which based on microelectrode impedance.This system had the advantage of automated data collection and real time analysis.To standardize the loading volume and time of administration for prebiotics evaluation with Caco-2 cell model based on RTCA SP system,we conducted the following experiments:(i)measure the effects of 3 different SCFAs mixtures on Caco-2 cells growth;(ii)determine the effect of fructo-oligosaccharide(FOS)and resistant starch fibosol-2(RS)fermentation supernatant on Caco-2 cells growth and Caco-2 monolayer barrier function.We found that the tolerance range of Caco-2 cells to SCFAs mixtures was 0-8 mmol/L.So we fixed the loading amount as 6.3%(v/v).Also,we found that the administration time range of Caco-2 cell growth model and Caco-2 monolayer barrier function model was 0-16 h and 0-72 h respectively.So data of 16 h in Caco-2 cell growth model and 72 h in Caco-2 monolayer barrier function model were picked up respectively as final results to compare FOS and RS.FOS and RS fermented with fresh fecal samples anaerobically in two nutritional conditions(PBS system and BCM system).RTCA SP system was then applied to evaluate the fermentation supernatant of FOS and RS with above experimental parameters.We found that two prebiotics stimulated Caco-2 cell growth and enhanced Caco-2 monolayer barrier function;Especially in PBS system,supernatant of FOS and RS(in fermentation of 24 h and 72h)promoted cell growth significantly.In order to confirm the validity of above evaluation,the abundance of key OTUs responding to prebiotics and the contents of SCFAs in the fermentation supernatant were analyzed.Results showed that physiological functions of FOS and RS fermentation supernatant were positively correlated with SCFAs-producing bacteria genera Faecalibacterium and Roseburia significantly and negatively with opportunistic pathogens such as Oscillibacter,Bilophila,Escherichia/Shigella and Streptococcus.Effect of FOS and RS fermentation supernatant on Caco-2 monolayer barrier function positively correlated with contents of acetic acid,propionic acid,butyric acid and total acids,and negatively correlated with iso-butyric acid.In summary,RTCA SP system was introduced to the evaluation of prebiotics fermentation supernatant and conformed the loading volume and time of administration of Caco-2 cell models in this system.Based on this physiological functions of FOS and RS fermentation supernatant were evaluated.The correlation analysis between physiological functions of FOS and RS fermentation supernatant and the change of microbial community structure of fermentation broth or the contents of SCFAs in the fermentation supernatant revealed that prebiotics stimulated Caco-2 cell growth and Caco-2 monolayer barrier function by modulating the community structure of fermented bacteria and affecting their metabolic products.This study indicated the reliability of the in vitro evaluation of prebiotics with RTCA SP system.